Various scientific and scholarly articles are referred to in brackets throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
Plant growth and biomass accumulation are dependent on the availability, absorption and assimilation of nutrients from the environment. Nitrogen is one of the principal factors limiting vegetative production. Only a few micro-organisms are capable of reducing molecular atmospheric nitrogen (N2) to a form usable by the plant. Plants themselves are unable to do this. Although some plants can utilize N2 through a symbiosis with certain nitrogen-reducing micro-organisms, the majority of plants obtain nitrogen by assimilating nitrate and ammonium from the soil.
In forest trees, the capacity to assimilate nitrogen is of particular significance. In communities of perennial, long-lived species, most of the nitrogen is fixed in living tissue and is not in the soil. The availability of inorganic nitrogen in the soil is a limiting factor to tree growth (Cole and Rapp, 1981, In Reiche DE (ed) Dynamic Properties of Forest Ecosystem, pp. 341-409, Cambridge University Press, Cambridge). Poplar trees are often used in reforestation efforts, and a variety that can grow well in low nitrogen soil would be very useful. Additionally, fast-growing, high biomase poplar trees are also very valuable for fodder and industrial wood production.
Assimilation of nitrogen by plants entails the reduction of nitrate to ammonium and its incorporation into carbon skeletons. Ammonium is assimilated into organic nitrogen mainly through the reaction catalyzed by glutamine synthetase (GS; EC 6.3.1.2). The amide group from the product of the GS reaction, glutamine, is then transferred to glutamate by action of glutamate synthetase (GOGAT; EC 1.4.7.1 and 1.4.1.14). This metabolic pathway is of crucial importance, since glutamine and glutamate are the donors for the biosynthesis of major nitrogen-containing compounds, including amino acids, nucleotides, chlorophylls, polyamines, and alkaloids (Miflin and Lea, 1980, In Miflin B J (ed) The Biochemistry of Plants Vol 5 pp. 169-202, Academic Press Inc., London).
The biochemistry and molecular biology of the GS/GOGAT cycle has been extensively studied due to the key role these enzymes play in plant growth and development (Crawford and Arst, 1993, Ann Rev Genet 27:115-146; Lam et al., 1996, Ann Rev Plant Physiol Plant Mol Biol 47: 569-593). GS is encoded by a small family of homologous nuclear genes. The enzyme is represented by two main isoenzymes: GS is localized in the cytosal and GS2 is a chloroolastic enzyme. Octameric GS holoenzymes also differ in their subunit compositions, GS1 is comprised of polypeptides of 38-41 kD in most plant species, whereas the size of GS2 subunit polypeptides is about 45 kD.
The physiological roles of GS1 and GS2 are now relatively well-established in angiosperms (Lea, 1997, In Dey P M, Harborne J B (eds) Plant Biochemistry, pp. 273-313, Academic Press, San Diego). In leaves, GS2 is expressed in photosynthetic tissues and is responsible for the incorcoration of ammonium from nitrate assimilation and photorespiration. GS1 as mainly expressed in vascular elements and functions to generate glutamine for nitrogen transport within the plant.
Approaches to modify levels of key enzymes involved in steps in carbon and nitrogen assimilation and primary metabolism have been considered as a means to improve vegetative growth and biomass production (Foyer and Ferrario, 1994, Biochem Soc Trans 22: 909-915, and the references therein). All the work on metabolic engineering of the nitrogen assimilation using transgeric plants has been done in annual, herbaceous species (e.g., tobacco, Lotus corniculatus L.). Increases in protein content and biomass production have been reported in transgenic tobacco expressing a pea GS1 gene (Coruzzi, 1995, International Application, Patent Cooperation Treaty, WO 95/09911).
However, success with constitutively-expressed GS transgenes has been unpredictable. In other reports, transgenic herbaceous plants which over-express cytosolic GS1 fail to exhibit changes in protein, chlorophyll or biomass production (Eckes et al., 1989, Mol Gen Genet 217: 263-268; Hirel et al., 1992, Plant Mol Biol 20: 207-218; Temple et al., 1993, Mol Gen Genet 236: 315-325). These discrepancies may be due to the instability of the holoenzyme in a heterologous system and/or to the different plant species used in the transformation studies.
The effects of GS over-expression may be unique in the woody perennial species, as compared to herbaceous annual species. Woody perennial species uniquely store assimilated nitrogen during periods of less favorable growth conditions, such as would occur in winter. In poplar, for example, assimilated nitrogen can reside as vegetative storage proteins (VSPs). VSPs can be mobilized to support development during active growth (Ryan and Bormann, 1982, BioScience 32: 29-32). Synthesis of seasonal VSPs in poplar is dependent on environmental factors, including photoperiod and nitrogen availability (reviewed in Coleman, 1993, in YWC N. B. Klopfenstein M-S. Kim, M. R. Ahuja, eds, Micropropagation, Genetic Engineering, and Molecular Biology of Populus, Gen. Tech. Rep. RM-GTR-297, pp 124-130, Rocky Mountain Forest and Range Experiment Station, Fort Collins). Since spring shoot growth in poplar is correlated with nitrogen recycling (Coleman et al., 1993, Plant Physiol 102: 53-59) and glutamine is the main amino acid transported in spring xylem sap (Sauter and van Cleve, 1992, Trees 7: 26-32), enhancement of nitrogen-use efficiency as a result of ectopic GS expression could affect the availability of reduced nitrogen for initiation of rapid spring growth.
The slow growth of woody perennials as compared to herbaceous annuals may also make the effect of GS over-expression on plant growth and physiology unpredictable. Because of this comparatively slow growth rate, a particular enzyme or metabolic pathway may influence plant growth and development may be quite different than that of fast-growing herbaceous plants. This may be especially true when targets for metabolic engineering are enzymes involved in assimilation and primary metabolism, therefore having wide-spread effects on plant development.